Various therapeutic methods and therapeutic drugs for malignant tumors have been developed, but sufficient therapeutic effects often have not been able to be obtained yet. Antineoplastic agents such as taxol, carboplatin and irinotecan are effective, but their side effects are strong, and thus, reduction of the side effect has been desired.
Meanwhile, HB-EGF is known to be a cell growth factor of the EGF family and as a molecule which is essential for formation and regeneration process of a body as well as is involved in occurrence of vascular stenosis and arteriosclerosis (see, e.g., Non-patent literature 1). This molecule is synthesized as a membrane bound precursor (proHB-EGF), and is cleaved on the cell surface with protease to produce the soluble form HB-EGF. A growth promoting action is observed in the soluble form whereas a growth inhibitory action is observed in the membrane-anchored form. Thus, HB-EGF seems to serve for formation and maintenance of tissues by appropriately using the soluble form or the membrane-anchored form as the situation demands.
HB-EGF is bound to EGF receptor (EGFR) (Her1) and Her4 (ErbB-4) in the EGFR family and activates them. However, members (Her1, Her2, Her3 and Her4) in the EGFR family can form heterodimers in all combinations as well as form homodimers. Thus, consequently, HB-EGF can activate all molecules in the EGFR family. HB-EGF is expressed in various tissues, and appears to act in broad cells and tissues, and is reported to promote well the growth of fibroblasts, smooth muscle cells and keratinocytes (see e.g., Non-patent literature 2).
HB-EGF is synthesized as the membrane bound precursor (proHB-EGF) as described above, and proHB-EGF is composed of, from an N terminus, a signal sequence, a prosequence, a heparin binding domain, an EGF-like domain, a juxtamembrane domain, a transmembrane domain and a cytoplasmic domain (FIG. 1). This proHB-EGF becomes the soluble form by being cleaved with protease (ectodomain shedding) at a portion indicated by an arrow in the figure. It has been proposed that the ectodomain shedding of proHB-EGF is stimulated by a pathway in which lysophosphatidic acid (LPA) activates Ras-Raf-MEK pathway through a G protein-coupled receptor or a pathway in which phorbol ester activates PKC (see e.g., Non-patent literature 3).
A function that the soluble form HB-EGF is bound to EGFR and facilitates phosphorylation of EGFR is present in the EGF-like domain (see e.g., Non-patent literature 1).
It has been known that diphtheria toxin is a protein having a molecular weight of about 59,000 produced by diphtheria bacillus and is bound to the membrane-anchored form precursor (proHB-EGF) of HB-EGF as the receptor (see e.g., Non-patent literature 4). Also, a mutant such as CRM197 of diphtheria toxin is known as an inhibitor of the soluble form HB-EGF (see e.g., Non-patent literature 5). Database information of diphtheria toxin is available for its gene in EMBL; K01722, its amino acid sequence in SWISS-PROT; P00588 and its three dimensional structure in PDB; 1MDT or 1×DT. A phage lysogenized in a diphtheria bacilli encodes the gene of diphtheria toxin.
Diphtheria toxin is a simple protein composed of 535 amino acid residues (the amino acid sequence [SEQ ID NO:1] of diphtheria toxin and a base sequence [SEQ ID NO:2] of the gene encoding it are shown in FIGS. 2 and 3, and italic letters represent the signal sequence). Diphtheria toxin can be separated into fragment A and fragment B by treating with a reducing agent (FIG. 4). According to conformational analyses, the fragment B is further divided into two domains. For the function of each domain, a catalytic domain corresponding to the fragment A (amino acid numbers 1 to 193 when the signal sequence is excluded) has an ADP ribosylation activity, a transmembrane domain (amino acid numbers 194 to 378 when the signal sequence is excluded) corresponding to an N terminal half of the fragment B has a nature to form a channel in an endosome membrane, and a receptor-binding domain (amino acid numbers 386 to 535 when the signal sequence is excluded) corresponding to a C terminal half of the fragment B has an activity to bind to a diphtheria toxin receptor on the cell surface.
The fragment A (catalytic domain) of diphtheria toxin has the action to ADP-ribosylate EF-2 (elongation factor 2) in the presence of NAD, thereby inhibiting protein synthesis. Therefore, in order to exert the toxicity of diphtheria toxin, the fragment A must enter in cytoplasm.
In the mechanism in which the fragment A enters in the cytoplasm, the receptor-binding domain in the fragment B is bound to proHB-EGF which is the receptor on the cell surface to internalize diphtheria toxin by endocytosis, then the transmembrane domain is inserted in the endosome membrane in the endosome, and finally the fragment A is released in the cytoplasm by passing through the endosome to inactivate EF-2 there (see e.g., Non-patent literature 6).
To exert the toxicity of diphtheria toxin, both the fragments A and B are necessary. Therefore, if either the fragment has a mutation, a protein having no toxicity of diphtheria toxin can be generated.
In diphtheria toxin, the detoxified mutant such as CRM197 having the mutation in the catalytic domain has been isolated.
Meanwhile, the mutant of diphtheria toxin has the activity to inhibit the binding between HB-EGF and EGFR because diphtheria toxin is bound to the EGF-like domain of the soluble form HB-EGF. The receptor-binding domain of diphtheria toxin is involved in this binding. It has been reported that Lys at position 516 and Phe at position 530 in diphtheria toxin are important for the binding to HB-EGF (see e.g., Non-patent literature 7). A crystal structure of a complex composed of diphtheria toxin and the EGF domain of HB-EGF has been analyzed, and the important amino acid residues for binding to HB-EGF have been reported to be between positions 381 and 535 (see e.g., Non-patent literature 8).
This way, it has been observed that diphtheria toxin mutant is bound to HB-EGF and inhibits the activity of HB-EGF. Recently, it has been attempted to use diphtheria toxin mutant as the therapeutic agent for the cancer by targeting HB-EGF for cancer therapy, but the attempt has not come into practical use yet (Patent document 1, Non-patent literature 9).
Patent document 1: JP 2004-155776-A;    Non-patent literature 1: Mekata E. et al, “Idenshi Igaku” Vol. 5, No. 2, P. 131-134, 2001, Medical Do Co., Ltd.;    Non-patent literature 2: Higashiyama, S. et al., J. Cell Biol., 122: 933-940, 1993;    Non-patent literature 3: Prenzel, N. et al., Nature 402: 884-888, 1999;    Non-patent literature 4: J. G. Naglich et al., Cell 69: 1051-1061, 1992;    Non-patent literature 5: T. Mitamura et al., J. Biol. Chem., 270: 1015, 1995;    Non-patent literature 6: T. Umata et al., J. Biol. Chem., 273: 8351, 1998;    Non-patent literature 7: Shen, H S et al., J. Biol. Chem., 269: 29077-29084, 1994;    Non-patent literature 8: Gordon V L et al., Molecular Cell 1: 67-78, 1997;    Non-patent literature 9: Miyamoto, S. et al., Cancer Res., 64: 5720-5727, 2004.